During its development a plant shoot progresses through a juvenile phase and an adult phase and then begins to produce reproductive structures. Recent screens for mutations that accelerate the juvenile-to-adult transition in Arabidopsis have produced three genes--ZIPPY (ZIP), SGS2/SDE1, and SGS3--that have nearly identical mutant phenotypes and which appear to operate in the same pathway/process. ZIP encodes an Argonaute protein, whereas SGS2/SDE1 and SGS3 encode, respectively, an RNA-dependent RNA polymerase and a novel protein. SGS2/SDE1 and SGS3 were originally identified as genes required for posttranscriptional gene silencing (RNAi), and are thought to be involved exclusively in this process because their effect on plant morphology has not been previously recognized. The identity of these genes strongly suggests that vegetative phase change is regulated by a pathway in which negative regulation, mediated by either miRNAs and/or siRNAs, plays a critical role. However, the location of this pathway (leaves, roots, shoot apical meristem?) and the genes that are direct and indirect targets of ZIP, SGS2, and SGS3 remain unknown. This information is critical for understanding the mechanism of vegetative phase change and the function of these three genes in normal development. We propose to determine where these genes function in the plant by constructing genetic mosaics using the GAL4 transactivation system. Genes regulated by ZIP, SGS2 and SGS3 will be identified by microarray analysis, by screening for mutations than suppress the phenotype of zip, and by determining if ZIP, SGS2 and SGS3 regulate the activity of SPL-like and AP2-like genes, two miRNA-regulated gene families that appear to play a role in vegetative phase change and floral induction. In addition, several newly identified genes that appear to operate in the same pathway(s) as ZIP, SGS2 and SGS3 will be characterized. It is becomingly increasing obvious that post-transcriptional gene silencing mediated by either miRNAs or siRNAs is required for many processes in plants, fungi, and animals and can be used in the therapy of diseases of viral and genetic origin. However, the mechanism of this process and its biological functions still remain to be determined. The research described here will reveal the role of PTGS in temporal regulation in plants, and will contribute to future studies of the mechanism of this process in both plants and animals.